1. Field of the Invention
The present invention relates to an enzyme electrode arrangement.
2. Description of the Related Art
Biomolecule detection is of key importance in pharmaceutical research and clinical diagnostics. Typically, this detection is performed utilizing a so called affinity reaction where complementary analytes bind together specifically. Fundamentally, there is the problem of detecting the smallest concentrations available in one test. Mostly, today's measurement systems sensitivities are too small to directly detect these low concentrations. Therefore, amplification schemes amplifying indirectly the signal as much as possible above the lower limit of detection of the measurement system are used.
Affinity sensors are a special type of biosensors utilizing any type of biomolecular recognition of highly specific affinity partners as e.g. antibody-antigen, nucleic acid-complementary nucleic acid, or receptor-ligand. In general bio- and affinity sensors are composed of two components: the biological component for the specific recognition of an analyte and the detector component, the so called transducer that should capture this biomolecular reaction and transform it into an analyzable signal. The selective biological components (e.g. nucleic acids, enzymes, antibodies, antigens or micro organisms) are intimately immobilized to the transducer and nowadays can be detected with a broad range of measurement principles. The choice of the transducer is governed by the reaction of the biological component and the resulting changes. The state of the art describes many transducers working on different principles. Common transducers are working on electrochemical, electrical or optical principles.
Sensitivity and specificity of the biomolecular recognition indicate—amongst other parameters like reproducibility, manufacturing costs, usability—the quality and practical usability of the sensor. The specificity of the biosensors is largely made-up by the biological component. One specific bioactive material at a time is used to detect the searched substance out of many others—also similar ones. The chemical reactions thereby taking place influence physical parameters, for example the electrical potential. The basic values or their changes respectively are transformed by the transducer in an electrical output signal that becomes subsequently electronically amplified. As a result, the biological component of the sensor, e.g. an enzyme, a receptor, or an antibody, binds the substance to be analyzed—essentially only this one—and generates a signal whose intensity is proportional to the concentration of the substance bound. For example, using enzymes the generation of a reaction product can generate an electrical signal that is picked up by an electrode. In the case of an enzyme sensor for the determination of glucose, the electrode is typically covered with a thin membrane comprising the immobilized glucose oxidase (GOD). The enzyme molecules are, e.g., entrapped in gelatin or polyurethane. The membrane is permeable only for molecules of a particular size. Out of a mixture of amino acids, proteins, fats, glucose, and other sugars the enzyme GOD converts only the glucose, also consuming oxygen. Glucono-lactone and hydrogen peroxide is formed. The immobilized enzyme is not washed out of the membrane but can be reused until its natural aging. In contrast, the smaller molecules like glucose, hydrogen peroxide and oxygen easily enter the membrane from the solution to be analyzed and vice versa. The hydrogen peroxide generated in the enzymatic reaction, as an electro active substance, donates two electrons per molecule to the electrode. The electrons generated in this reaction are subsequently delivered to the biosensors electrode—generating a micro-current. Simultaneously to the reaction of the glucose with the enzyme GOD, sometimes on a bio-inactive field, a blank current is measured that can be generated by interfering substances in the blood sample. Such interfering substances can be for example drugs, vitamins, or metabolic products in high concentrations that can also generate a micro-current. The net-current calculated in this way is a measure for the amount of glucose in e.g. a blood sample and can be converted into a proportional blood glucose concentration.
Difficulties still pose problems such as being reproducible, simple to use, and stable immobilization of the biological component. For example, to obtain a fast response time and a reliable reading a thin layer of immobilized bio-molecule is desirable and shelf life and operational stability demand a high value of immobilized enzymatic activity. Adsorption to adequate surfaces including a metal electrode layer yields relatively unstable systems. The associated problems are aggravated with planar systems or carriers respectively, insofar as a basically mechanical sandwich set-up for making of a sensor can not be used anymore. In case of loss of the mechanical integrity of such sandwich type biosensors due to cracks formed in the individual membrane layers, the resulting readings are falsified due to the above mentioned interfering substances to an extent that even the above mentioned differential measurement method using a bio-inactive field cannot compensate.
Therefore, the object underlying the present invention is to provide a stable enzyme electrode arrangement or biosensors respectively, made in a sandwich set-up, in order to enable fast and highly precise measurements without the influence of interferences during continuous use and in this way provide a reliable signal amplification of biological binding reactions. This can be realized without covalent coupling of the biological component typically immobilized within a membrane.